**E** ff**ect of Salt Addition upon the Production of Metabolic Compounds by** *Yarrowia lipolytica* **Cultivated on Biodiesel-Derived Glycerol Diluted with Olive-Mill Wastewaters**

### **Markella Tzirita 1, Maria Kremmyda 1, Dimitris Sarris 1,2, Apostolis A. Koutinas 1 and Seraphim Papanikolaou 1,\***


Received: 21 August 2019; Accepted: 16 September 2019; Published: 24 September 2019

**Abstract:** One of the major environmental problems is the highly toxic agro-industrial waste called olive mill wastewater (OMW), deriving from olive oil production. On the other hand, the continuous development of the biological liquid fuel industry (biodiesel and bioethanol) makes it mandatory the process and exploitation of their main by-products, crude glycerol. This study dealt with the biotechnological conversions of biodiesel-derived crude glycerol with the use of the non-conventional yeas<sup>t</sup> *Yarrowia lipolytica* in media that had been diluted with OMWs. OMWs, employed as simultaneous liquid medium and substrate, is a new trend recently appearing in Industrial Biotechnology, where value-added metabolites could be produced with simultaneous partial detoxification (i.e. decolorization and phenol removal) of the used residue. In the present study, diluted OMWs (containing 2.0 g/<sup>L</sup> of total phenolic compounds) blended with 70.0 g/<sup>L</sup> crude glycerol were employed as substrates. Production of value-added compounds by *Y. lipolytica* strain ACA-YC 5031 was studied in nitrogen-limited media favoring the production of secondary metabolites (i.e. citric acid, polyols, microbial lipids, polysaccharides). Batch-flask cultures were carried out and the impact of the addition of di fferent NaCl concentrations (1.0%, 3.0%, 5.0% *w*/*w*) added upon the biochemical behavior of the strain was studied. Remarkable biomass production was observed in all trials, while in the "blank" experiment (no OMWs and no salt added), the metabolism was shifted toward the synthesis of polyols ( <sup>Σ</sup>polyols = mannitol + arabitol + erythritol > 20 g/<sup>L</sup> and maximum total citric acid-Cit (sum of citric and isocitric acid) = 10.5 g/L). Addition of OMWs resulted in *Citmax* = 32.7 g/L, while <sup>Σ</sup>polyols concentration dropped to <15 g/L. Addition of salt in the OMW-based media slightly reduced the produced biomass, while *Cit* production drastically increased, reaching a final value of 54.0 g/<sup>L</sup> (conversion yield of Cit produced per unit of glycerol consumed = 0.82 g/g) in the trial with addition of 5.0% NaCl. Finally, significant color and phenols removal were observed, evaluating the yeas<sup>t</sup> as a decontamination medium for the OMW and a grea<sup>t</sup> candidate for the production of value-added compounds.

**Keywords:** Biodiesel-derived glycerol; citric acid; microbial lipid; olive-mill-wastewater; polyols; polysaccharides; *Yarrowia lipolytica*; value-added compounds
